Hi,

It sound a bit crude to me. Have you purified the thymocytes, or are there still Tcells, Bcells, Granulocytes, Endothelial cells, stromal cells etc. present?

I don't think you can get antibodies specificially directed against tymocytes. Likely you will get antibodies against a lot of different antigens.

Kerstin

There is no reason to obtain 90% of dead cells. Use fresh obtained human tissue, from an HIV negative and Hepatitis B and C negative person. You can gently grind or tease the thymus in the presence of RPMI or MEM media without será. Once you have the cell suspensión rapidely separate debris, and tissue from cell suspensión by short and slow centrifugation or wait to the appearence of a sediment. Now check cell viability and this crude cell suspensión will generate anti-thymocyte immunoglobulin. This type of crude  preparations were used many years ago exactly for that purpose . It is true that these crude cell contain mainly T cells but there are B, dendritic and monocyte as well as Kerstin mentioned

Dear Dr. Mario  and Dr. Remi,

Than q so much for your suggestion, I am expecting some more valuable guidelines and suggestion to sort some  practical problems and clarify certain doubts from you

I am getting  thymus organ from hospital after surgery ,  soaking in  (PBS +2.5% DMSO, as per the few literature study) and preserving the tissue sample in -80°C for long term storage. During the time of my requirement, I m taking out the thymus tissue from the freezer  and isolating the thymocytes by mechanical method (as what u explain in your answer part)  with albumin rich buffer: PBS (1X)+2mM EDTA+ 20% FBS+BSA.  I tried with RPMI and DMEM media, I observed cells are getting lysis, or reputered immediately, but I got good response in albumin rich buffer.

            After isolating the thymocytes, I am checking the cell viability with 0.08% Tryphan blue (100µL dye+100 µL ), I am getting 90 to 95 % cells are dead only, I have repeated this experiment with different thymus organ which  I, preserved in -80°C.  But with out dye when I am observing the cells under microscope, I am getting good number of cells, morphologically they good, intact and healthy.

Can you suggest me; (1) how to sort it out this kind of problem? Really those are dead cells ? (2) Can I use those cells for development of anitiboides against thymocytes in animal? (3) Is it the right way to store the thymus organ after collection from the hospital?

            In addition to that, debris and unwanted cells from the entire cell suspension were removed by sedimentation, further , I am pelleting down the cells @1500 rpm , no doubt I am getting tight pellet at the bottom of centrifuge tube, further, I m resuspending the pellet with fresh buffer according to my desired volume, but I found that the pellet is not getting disperse in the fresh buffer and some gelly kind of things are floating in the cell suspension, and cell number also I m getting very less, can you suggest me why its happening and how to sort it out?

Dear Sudhanson. PBS with DMSO is not good to preserve living cells. To answer your three questions: (1) There are several protocols to freeze cells.and preserve cells. Do not worry, mortality in many of those protcols even though is neve higher then 35% is always present,if you thaw the frozen vials correctly and follow the specific instructions you will improve cell survival. It is alwas necessary to use a culture media enriched with serum among other components. BUT do not worry for the (2) question answer is YES you can use your suspensión to immunize and boost animals to obtain anti thymocyte serum. (3) You can use the preserving cell techniques by freezing sells in suspension. BUT certainly not in the complete intact organ. Overcrowded cell suspensions form big clusters specially if there is calcium available in the liquid media. Timing in freezing and thawing is extremly important to preserve cell viability in all protocols. Freeze the cell suspensión NOT the whole organ and there is no need to use BSA because this soluble antigen for sure will stimulate anti BSA antibody production that may interfere in your experiments. Good luck let me know how this result.

Dear  Dr. Mario Sir,

Than q so much.  As per your Instruction, I have performed this isolation of thymocytes from fresh human thymus and its working well, but Sir , I got some technical problem, I hope u will clarify it for me as usually like previous time, Here , I have three question, please share your opinion  or any comments on my doubts.

             I took fresh thymus and Isolate the entire thymocytes by using RPMI media with mechanical method, I  got huge number of  live cells (trypan blue study), I preserved the cells by using 10% DMSO with RPMI media having 10% FBS and stored in -80C°, but after 48 hr. I, observed that, 80 to 90% cells are dead and I able to recover only 10% live cells. I repeated this experiment with other fresh thymus, even  though I got same kind  of problem .

1.Q  can u suggest me why its happening like that? How to get good quality of thymocytes?  Without staining, if u observed the cells under microspe, its looking morphologically very active, health cells only.

 2.Q. Those isolated dead cells can I inject to animal (either horse or rabbit) for development of antibodies against thymocytes?

3.Q. After isolating the cells from thymus, we are pipeting out cell suspension with pasture pipette and pelleting down the cell suspension at low rpm, and I am getting all the cells in the form of a pellet at the bottom, but I observed that, when I m trying to resuspending the pellet with fresh media, the tight pellet is not getting dispersed properly and some jelly kind of things are floating. Can u suggest me what could be the possible reason?

 Please Sir give little high priority in my question no. 2 basically,

Here without staining the cells, all r looking very health and active and no lysis but after staining with dye i m getting huge number of dead cells, can i inject those cells (dead) to the animal? Is it beneficial

Please sir, if possible, share your email id  with me.

Than q Sudhansu

Dear Sudhansu

One of the reasons for high mortality after freezing may be related to high cell density, criovials can hold one or two milliliters with low to medium cell density. Decreasing cell density will improve your success. 2 Question: YES YOU CAN INJECT THE DEAD CELLS AND LIVE CELLS MIXED to obtain excellent high concentration anti tynmocytes antibodies.. To prevent or mmini ize cell clothing first AVOID calcium from your media and decrease speed and centrifugation time,  Good luck

Dear Dr. Mario Cesar

As per your instruction, I started Injecting the human thymocytes to the rabbit,  now they  are under immunization state. very soon I m going to collect the blood samples from animal.  Please clarify my two doubts related to my experiment.

 Q1. Can u suggest me what is the best method for isolation and purification of human antithymocytes immunoglobulin (polycolonal antibodies) from rabbit serum?

After complete the process of isolation and purification

, Q2. what are the assays I need to perform to confirm the purified antibodies were  antithymocyte immunoglobulin only?

Please suggest me several methods for purification and isolation of polyclonal antibodies from rabbit serum,  I will perform simultaneously to get better and accuracy result.

Can u share your email id to me

Dear Sudhansu. Depending the quality of purification you can try two differents techniques. A Classical Cohn etanol cold precipitations briefly as follows: 1 let fresh blood from hyperimmunized rabbits to clot, remove clot (this mainly fibrinogen or fraction I), 2, Add drop by drop cold etanol to obtain a precipitate qhich is almost immunoglobulins this is called fraction II and was used by many multinational companies around the world for antibody isolation for medical aplications. The other technique is also simple: 1) Ammonium sulphate precipitation, once you prepared the ammonium sulphate saturated solution, add drop by drop under cold conditions (use ice) to obtain 50 % saturation and you will obtain a precipitate, discard the supernate and resuspend the precipitate with saline solution and precipitate again with the same saturated solution to obtainNOW A 33 % SATURATION, remove supernate and suspend the precipitate with saline solution. IT IS NECESSAR then to dialyze to remove the used salt. Using either of these two techniques you have now anti human thymocyte hiperimmune globulin from rabbits. Additional purification can be obtained to remove IgM or to isolate only IgG using DEAE cellulose. Regardin to Q2 You can use a simple test of mixing ten microliters of your purified globulin and one hundred thousand fresh human thymnocytes in 0.5 ml of RPMI , add fresh human serum as source of fresh Complement and incubate for 1 hour at 37 celsius, use trypan blue exclusion test to quantify colored dead cells with and without your globulin preparation. Include appropriets controls.Good luck

Dear Dr. Mario

Than q so much for your valuable suggestion, Sir , with the reference to my previous messages, you have suggest some preliminary experiments (tryphan blue study) to confirm the isolated antibodies will be against human thymocytes only (antithymocytes immunoglobulin).

 In addition to that can u suggest me what the other scientific methods available that I need to perform for further confirmation?

Q.2. Human Anti thymocyte globulin ELISA Kit are available commercially in the market, can we use those kits or is it the right kits to check the quality and quantity of antithymocytes immunoglobulin antibodies after isolation from rabbit serum

Dear Dr. Mario

I have given all the doses (human thymocytes) to the rabbit, Now I am collecting the rabbit serum, Can u tell me in a descriptive manner once again   Q.1. How I will estimate the ATG (Antithymocyte globulin) in the rabbit serum?  Q2. What are the methods that I need to perform to check the antibody titre value in rabbit serum to confirm weather the antibodies against human thymocytes raised or not? is there any ELISA kits available to check quantitatively the antibody concentration in the collected serum? Sir this time please mentioned the protocol in a descriptive manner.